The vertebrate spinal cord contains a central pattern generator (CPG) capable of producing coordinated locomotion (e.g., walking, swimming, flying), even in isolation from the brain. However, the neuronal populations that generate the locomotor rhythm are unknown. Identifying the neural populations that generate locomotion would remove a major barrier in the study of the modulation, degeneration and regeneration of locomotion. The objective of this application is to characterize one promising population of spinal neurons: the V3 interneurons. The V3 neurons satisfy two important criteria for locomotor rhythm generation: first, they are located in the ventral medial spinal cord and, second, they are glutamatergic. We hypothesize that V3 neurons are core components of the locomotor CPG. In order to test this hypothesis, we propose three aims. First, we will determine the activity and recruitment patterns of V3 neurons during fictive locomotion. Next, we will determine if V3 neurons have locomotion generating membrane properties. Finally, we will determine if the V3 neurons are necessary for normal locomotion. The completion of these three aims will give us a better understanding of the role of the v3 neurons in locomotion and the overall structure of the vertebrate CPG.